Quantitation of the major whey proteins in human milk, and development of a technique to isolate minor whey proteins

NUTRITION RESEARCH, Vol. 8, pp. 853-864, 1988 0271-5317/88 $3.00 + .00 Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

QUANTITATION OF THE MAJOR WHEY PROTEINS IN HUMANMILK, AND DEVELOPMENTOF A TECHNIQUE TO ISOLATE MINOR WHEY PROTEINS Leslie R. Woodhouse, M.S. and Bo LSnnerdal, Ph.D. 1 Department of N u t r i t i o n , University of California, Davis, CA 95616 USA

ABSTRACT

Several whey proteins of humanmilk have important nutritional and physiological roles for the breast fed infant. Only 75 to 85% of the proteins in whey have been identified and quantitated, leaving a remaining protein fraction containing many proteins of probable nutritional and physiological significance. In this study, colostrum and mature milk samples were collected, and the concentrations of five major whey proteins were measured using immunoelectrophoresis or immunodiffusion. Based on Kjeldahl nitrogen analysis, the total concentration of whey proteins in colostrum was 17.05 mg/ml and in mature milk 5.95 mg/ml. The concentration of ~-lactalbumin was 3.7 mg/ml and 1.7 mg/ml in colostrum and mature milk, respectively; lactoferrin concentration was 7.3 and 1.5 mg/ml; and secretory IgA concentration was 4.7 and 1.2 mg/ml. In both colostrum and mature milk, concentrations of serum albumin and lysozyme were 0.37 and 0.07 mg/ml, respectively. The sum of these major whey proteins expressed as a percentage of the total whey protein accounted for 81% of mature whey protein, and 94% of colostral whey protein, leaving 19% and 6% in the remaining protein fraction. A method was developed to isolate this remaining protein fraction from the major whey proteins using gel f i l t r a tion and immunoaffinity chromatography. The quantitatively minor whey protein fraction obtained was further characterized using polyacrylamide gel electrophoresis and ion exchange chromatography. Key Words: humanmilk, whey proteins, immunochemistry, immunoaffinity chromatography. INTRODUCTION The superiority of humanmilk for promotion of infant health and development is well recognized (1,2). Humanmilk meets the protein requirement for infants, even though the protein content is quite low, 0.8 to 1.0% (3,4). The protein compartment contributes to many of the important physiological functions associated with breast feeding, including host resistance (5), high digestibili t y (2), and growth modulators (6). Since these important physiological functions have been associated with the protein component of humanmilk, i t is necessary to determine the correlation of the individual milk proteins to specific functions. This necessitates generation of knowledge of the protein fraction and its individual constituents, including identification, quantitation, and biological role. Corresponding author: Dr. Bo LSnnerdal, Department of Nutrition, Univ. of California, Davis, CA 95616 USA. 853

854

L.R. WOODHOUSEand B. LONNERDAL

The majority of human milk protein is found in the whey fraction (7), and several major whey proteins have been extensively studied (8,9,10). These proteins include ~-lactalbumin, lactoferrin, secretory IgA (sIgA), serum albumin, and lysozyme. Compiling data from the literature (11), these five proteins would comprise approximately 75% of mature whey proteins and 85% of colostral whey proteins. The remaining protein fraction includes a multitude of proteins with various functions, including enzymatic a c t i v i t y (12), binding properties (13,14, 15), or growth modulators (6), besides being a source of amino acids. Many of these proteins have only been shown to be present based on enzymatic a c t i v i t i e s or binding properties, but remain to be isolated, quantitated, and further characterized. I t is l i k e l y that within this remaining fraction, with better quantitation and characterization, several more whey proteins with presently unknown functions w i l l be found. The term major whey proteins needs to be defined, because i t is not used in a s t r i c t l y quantitative sense. There are several whey proteins identified that are found in humanmilk in higher concentrations than that of one of the major whey proteins, lysozyme (10). Lysozymecontent varies from 0.05 mg/ml to 0.25 mg/ml (16,17), i t has been well characterized (18), and a bacteriostatic role in the infant has been suggested (7). In contrast, the concentration of free secretory component that is normally associated with slgA, has been found at levels of 0.2 to 0.3 mg/ml in humanmilk (19), but a role for secretory component in unbound form has only been proposed, and longitudinal studies are lacking. Evidence exists which supports the hypothesis that secretory component confers to IgA additional resistance to proteolytic degradation (20,21,22). I t has also been proposed by Brandtzaeg (23) and Heremans (24) that free secretory component may mediate transport of IgA and IgM across the mucosal epithelium. Two other humanmilk proteins, b i l e - s a l t stimulated lipase and corticosteroidbinding protein, have also been identified in human milk in estimated concentrations similar to lysozyme (25,16). Specific roles for these proteins in the breast fed infant have been proposed, but in vivo evidence and longitudinal studies are lacking. Accurate q u a n t i t a t i v ~ - m ~ d s have also not been developed for these minor proteins. The major whey proteins as defined here can all easily be measured accurately using immunochemical techniques. In order to quantitatively determine the fraction of the whey protein that remains to be studied, i t is necessary to quantify those major whey proteins whose functions have been elucidated. The development of a technique to isolate the quantitatively minor whey fraction w i l l enable further characterization. Through the use of immunological methods, we have quantified the major whey proteins in mature milk and colostrum. By using gel f i l t r a t i o n and immunoa f f i n i t y chromatography, we have specifically eliminated the major whey proteins, obtaining a whey protein fraction that can be further characterized. MATERIALS AND METHODS Sample collection Mature milk samples (more than 1 month postpartum) or colostrum samples (days i-4 postpartum) were collected from healthy donors using a manual breast pump (Gentle Flow, Eklund & Co., Davis, CA}, or by hand expression. Aliquots of the whole milk were taken for Kjeldahl nitrogen analysis and the remainder was ultracentrifuged fresh at 189,000 x g for I h at 4~ The fat layer was removed and the supernatant (whey~ was aspirated and stored at -18~ in plastic, acid washed vials.

ISOLATION OF HUMANWHEYPROTEINS

855

Protein analysis Total nitrogen and non-protein nitrogen were determined using microKjeldahl analysis (26) with sample volumes of 1.0 ml (Kjeltec Auto 1030 Analyzer, Tecator, Hoganas, Sweden). To determine non-protein nitrogen, 1.0 ml of 24% trichloroacetic acid was added to 1.0 ml of whey, followed by centrifugation at 10,000 x g for 15 min at 4~ The supernatant, which contains the nonprotein nitrogen, was then analyzed for nitrogen. To obtain a value for protein nitrogen, non-protein nitrogen was subtracted from total whey nitrogen and multiplied by 6.25 for conversion of nitrogen to protein. Individual whey proteins were analyzed by immunoelectrophoresis or immunodiffusion (27,28). Lactoferrin, sIgA, and serum albumin were determined by rocket immunoelectrophoresis according to Laurell (29), using monospecific antibodies to human lactoferrin, slgA, and serum albumin (Accurate Chemicals, Westbury, NY). Purified lactoferrin was obtained according to the method of Davidson and LSnnerdal (30). Purified slgA and human serum albumin were purchased from Calbiochem-Behring (La Jolla, CA). Lysozymeand ~-lactalbumin were determined by single radial immunodiffusion according to Vaerman (31). Lysozyme and ~-lactalbumin antibodies were purchased from Dakopatts (Santa Barbara, CA). Purified ~-lactalbumin was also obtained from Dakopatts, and purified lysozyme was prepared using cation exchange chromatography (32). Modification of the agarose (Bio-Rad, Richmond, CA) for improved immunoelectrophoresis was performed according to the method of LSnnerdal and L~s (33). Gel f i l t r a t i o n chromatography Whey samples (1 ml) were chromatographed on a Sephadex G-75 Superfine (Pharmacia Fine Chemicals, Piscataway, NJ) column (1.2 x 50 cm) in 0.1M ammonium acetate, pH 6.5, with a flow rate of 12 ml/h in order to separate the low molecularweight proteins ~-lactalbumin and lysozyme. Fractions were monitored for absorption at 280 nm. Immunoaffinity chromatography Immunoaffinity chromatography (35) was used to specifically remove several major whey proteins from whey samples. Monospecific antibodies against the individual whey proteins lactoferrin, slgA, and serum albumin were separately immobilized on CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ). The basic coupling procedure was the same as that described by Pharmacia (36). The gel was washed with coupling buffer (0.1M NaHCO:, pH 8.3 and 0.5 M NaCl) and combined with the antibody (ligand) in a concentration of approximately 5 mg/ml gel. The immunosorbentswere packed in I x 5 cm columns, and following the coupling, the remaining active groups were blocked with the addition of 0.2 M glycine-HCl, pH 8. Following washing, the immunosorbentwas equilibrated with the immunoaffinity chromatography buffer, 1M Tris-HCl, pH 8 + 0.5 M NaCl, to avoid non-specific adsorption. A flow rate of 0.5 ml/min was used for the column runs, and fractions were monitored for absorption at 280 nm and collected. The antibody-bound antigen was desorbed with 0.5 M acetic acid, pH 2 (2 ml). These immunosorbent columns were used repeatedly with retained antigen-binding capacity. Polyacrylamide 9el electrophoresis A slight modification of the acetic acid-urea polyacrylamide gel electrophoresis system described by Smith (37) was used. All reagents and supplies used in the electrophoresis were purchased from Bio-Rad (Richmond, CA), unless otherwise noted. A T7C2.5 acrylamide running gel and a T5CI.5 stacking gel were

856

L.R. WOODHOUSEand B. LONNERDAL

used. The gels contained 2.5 M urea, and pyronin Y (Sigma Chemical Co., St. Louis, MO) was used as the tracking dye. All samples were dissolved in a sample solvent of 10% I M HCI and 5.4% (w/v) urea in deionized water. Electrophoresis was run at 2 mA/gel f o r 4-6 h (depending on sample migration) in 0.9 M acetic acid. Gels were stained f o r 3 h in a s o l u t i o n of 0.1% (w/v) Coomassie B r i l l i a n t Blue R250, 50% methanol, 40% deionized water, and 10% g l a c i a l acetic acid, followed by destaining in a solution of 10% g l a c i a l acetic acid, 10% methanol, and 80% deionized water.

Ion exchange chromatography Ion exchange chromatography using FPLC (fast protein liquid chromatography) (Pharmacia Fine Chemicals, Piscataway, NJ) allowed rapid separation of whey proteins. The ion exchanger beads (Mono Q) have a very uniform and rigid structure which results in separations of very high resolution. A 0.02 M ethanolamine, pH 9.5 buffer using a linear ionic strength gradient to i M NaCl was used for the anion exchange, with a flow rate of 0.5 ml/min. Fractions were monitored at 280 nm. Whey samples were diluted 1:5 with starting buffer, 500 ~l was applied, and the run was monitored at a sensitivity of 0.20D (full scale). More dilute protein solutions from the immunoaffinity chromatography runs were dialyzed i n starting buffer, 500 ul was applied, and the run was monitored at a sensitivity of 0.02 OD (full scale). RESULTS Protein content Kjeldahl nitrogen analysis of the colostrum and mature milk samples is shown in Table I . Individual whey protein concentrations are shown in Table 2. Radial immunodiffusion was chosen to q u a n t i t a t e ~-lactalbumin and lysozyme because they have molecular weights of approximately 13,000-15,000 and single

radi~l immunod~ffusion quantitates proteins in a wider mol~cular weight range (5 x 10 - 2 x 10"') than rocket immunoelectrophoresis !3 x 10~ - 2 x 10 ) (29). Commercially purified Standards were available for some milk proteins but not a l l . Column chromatography techniques were developed and used to isolate and purify lactoferrin and lysozyme. The protein purity was determined using polyacrylamide gel electrophoresis, and the protein content was analyzed by the Lowry protein assay. Table 3 shows mean individual protein concentrations and total protein as determined by Kjeldahl analysis. The sum of the total major whey proteins in mature milk accounted for 80.8% of the total whey protein, leaving a 19.2% remaining protein fraction. The sum of the total colostral whey proteins accounted for 94.5% of the total whey protein, leaving a 5.5% remaining protein fraction. Figure I depicts the approximate breakdown in individual protein content in colostrum and mature milk. TABLE i. Kjeldahl Nitrogen Analysis (mg/ml) Colostrum n=3 3.29 +- 0.34

Total N 3.23 • 0 . ~ Whey N 0,50 • 0.10 NPN Kjeldahl whey protein (whey N - NPN) x 6.25 17.05 -+ 2.92 A l l values are shown as x -+ SD

Mature Mil n=7 1.54 • 0.16 1.20 • 0.27 0.31 • 0.04 5.95 • 1.00

ISOLATION OF HUMANWHEY PROTEINS

857

TABLE 2. Concentrations of Individual Whey Proteins (mg/ml) Lactoferrin

Secretory IgA

Serum Albumin

Lysozyme

1.4] 1.68 1.70 1.51 1.17 1.62 1.28

0.93 0.54 0.96 1.60 0.32 1.41 2.2?

0.58 0.59 0.41 0.25 0.31 0.34 0.16

0.12 0.16 0.10 0.04 0.03 0.01 0.05

1.48 • 0.19 1.15 _+ 0.62 0.38 • 0.15

0.07 • 0.05

~-!actalbumin Mature 1.60 1.98 1.93 1.88 1.24 1.57 1.91

1 2 3 4 5 6 7

1.73 +- 0 . 2 5

~• Colostrum

2.58 4.29 7.00 2.50 I . 61 2.70 5.03

1 2 3 4 5 6 7

3.67 • 1 . 7 4

R • SD

8.06 5.22 7.08 7.43 9.02 7.47 6.66

4.89 3.24 9.30 NA 5.14 3.95 1.72

0.28 0.56 0.21 0.45 O. 39 0.32 O. 34

0.II 0.05 0.11 0.21 O.01 0.01 0.02

7.28 -+ 1.09 4.71 +_ 2.34 0.37 • 0.11

0.08 • 0.07

TABLE 3. Protein Content of Whey (mg/ml)

m-lactalbumin Lactoferrin Secretory IgA Serum albumin Lysozyme

Colostrum Whey n=7

Mature Whey n=7

3.67 7.28 4.71 0.37 0.08

1.73 1.48 1.15 0.38 0.07

• • • • •

1.74 1.09 2.34 1.06 0.07

• • • • •

0.25 0.19 0.62 0.15 0.05

16.11

4.81

17.05 • 2.92

5.95 • 1.00

Major protein fraction

94.5%

80.8%

Minor protein fraction

5.5%

19.2%

Total Kjeldahl protein

All values are shown as R • SD

L.R. WOODHOUSEand B. LONNERDAL

858

MATURE WHEYPROTEINS

COLOSTRI~ WHEYPROTEINS lysozyme .5~ serum albumin 2%

minor proteins 6g

minor proteins lgg

aipha-lactatbumin 22~

aipha-lacta}bumin

lysozyme 2% serum albumin 6%

secretory IgA

lactoferrin

27%

43~

secretory IgA

FIG. I. Protein content of mature whey and colostrum whey. Isolation of minor whey protein fraction The results of the Sephadex G-75 chromatography show that lysozyme and ~-lactalbumin (V~ = 37-42 ml) were well separated from the other whey proteins due to the large=difference in their molecular weights (Figure 2). The molecular weight of lysozyme is 15,000 and that of ~-lactalbumin is 14,100, while v i r t u a l l y all other identified whey proteins have molecular weights above 40,000 (10). Both peaks recovered were qualitatively tested for ~-lactalbumin and lysozyme using single radial immunodiffusion, and both proteins were present in the second peak and absent in the f i r s t peak.

Io0

--

A280 0.5 0

1 A I I.Oml whey

20

I 60

40

ELUTION VOLUME, ml

FIG. 2. Gel f i l t r a t i o n chromatography of mature whey (1.0 ml) on Sephadex G-75 Superfine in 0.1M ammoniumacetate, pH 6.6.

ISOLATION OF HUMANWHEYPROTEINS

1.0

A2B 0

859

/

B

/

0.5

0.5 M acetic ocid, pH2.0

I

o

f

5

Somple opplicotion

I0

,la.

15

EO

ELUTION VOLUME, ml

FIG. 3. Immunoaffinity chromatography of lysozyme and ~-lactalbumin-free whey on CNBr-activated Sepharose coupled with anti-lactoferrin in 1.0 M Tris, pH 8.0 + 0.5 M NaCl. For isolation of the three remaining major whey proteins, lactoferrin, sIgA, and serum albumin, a 3.0 ml fraction from the f i r s t Sephadex G-75 peak was applied to the anti-lactoferrin, column. All the lactoferrin became specifically bound to its immobilized antibody and therefore was removed from the sample. The 0.5 M acetic acid, pH 2 application desorbed lactoferrin (Figure 3). Again, qualitative analysis on the peaks was performed using immunoelectrophoresis, and i t was found that lactoferrin was only detectable in the second small peak (Vo:17-19 ml). Isolating the f i r s t peak and applying i t to the anti-slgA column resulted in removal of slgA, and isolation of the slgA-free peak applied to the anti-serum albumin column eliminated serum albumin. The final whey fraction obtained was devoid of all five previously mentioned major whey proteins, as confirmed by rocket immunoelectrophoresis. Polyacrylamide gels of the step-by-step elimination of the individual whey proteins are shown in Figure 4. With each successive column run, dilution occurred, resulting in increasingly fainter protein bands. The FPLC Mono-Q anion exchange chromatography runs are shown in Figure 5. The l e f t chromatogram is from the starting whey sample, the right chromatogram is from the prepared whey sample devoid of all five major whey proteins.

L.R. WOODHOUSEand B. LONNERDAL

860

1

2

3

4

5

;!;i!!!iiiiiiiiil;

iiiiiii;;i~i!i!i;i

:;iiii~;i~iCi

m~:!:~m::"~

FIG. 4 Polyacrylamide gel electrophoresis of whey fractions.

i!!i!ii!!!i!:.:!

ii~!}~

!i'iiiii~:i:;!:i~

i ~g!giii!!i~

iiii!!;~i[g;ii?il;

Whey ~-LA and lys-free whey ii!~::i!i}H t "! e-LA, lys and Lf-free whey ~-LA, lys, Lf and slgA-free whey i!!!!!!!iiiiiii ii 5. ~-LA, lys, Lf, slgA and SA-free whey

:~u::;~:F:::

1. 2. 3. 4.

:::::::::::::::::::::

WHEY

MINOR WHEY PROTEINS

2

A

i

1.0

.02

A

ew

<(

it89 0

0 20

0 ELUTION VOLUME, ml

7

./

/

8

0 20

0 ELUTION VOLUME, ml

FIG. 5. FPLC anion exchange chromatography of mature whey and minor whey proteins in 0.02 M ethanolamine, pH 9.5, with a linear ionic strength gradient to I M NaCl.

ISOLATION OF HUMANWHEYPROTEINS

861

DISCUSSION In this study the major whey proteins in human milk were quantitated using immunological methods, and i t was shown that these proteins comprise approximately 95% of total whey proteins in colostrum and approximately 80% in mature milk. These percentages are consistent with other studies (11,7), in which percentages of approximately 80% were estimated. An important comparison of our data with other studies is that we analyzed the individual protein contents of each milk sample and compared the total to i t s total protein content, resulting in a more accurate percentage of major proteins. Other studies have reported means of individual protein concentrations analyzed from different milk samples, and compared the sum of these values with total mean protein content of other milk samples. Since there is considerable variation in concentrations of whey proteins among women, the use of mean values can lead to misinterpretations. The use of Kjeldahl protein as a basis with which to compare individual protein data is based on the fact that Kjeldahl nitrogen of whey, when corrected for non-protein nitrogen, correlates well with values for protein content obtained from amino acid analysis (38). We found a large variability among the milk samples for the individual proteins. Lysozyme in particular had a range of 0.01 mg/ml to 0.16 mg/ml. Such a wide range in concentration has been previously reported (16,17). One observation made was that fresh milk samples analyzed for lysozyme generally had a lower lysozyme content than samples that had been frozen prior to assay. Goldman et al. (17) found the opposite effect when looking at the effects of cold storage on lysozyme. With a 24 h storage at 4~ of fresh milk, lysozyme content decreased 40%. However, the effects of freezing were not measured. There is a possibility that lysozyme aggregates with several other proteins upon storage at -18%, and this would cause an overestimation of lysozyme content in the previously frozen samples. This effect of storage on protein concentration was not found for any of the other proteins.

The technique described here for preparation of whey samples devoid of the major proteins can be a useful technique to isolate the multitude of remaining whey proteins for further characterization. The FPLC technique shows promise for protein analysis of human whey. The Mono-Q anion exchange column separated the minor protein fraction into several distinguishable peaks. By increasing the sample size applied, i t should be possible to increase peak sizes and to isolate those peaks for further identification and characterization. This separation method was found to be very fast (30 min/run) and highly reproducible. In summary, the major whey proteins of human milk have been extensively studied, and accurate quantitation methods exist. Based on our results and previous studies, the five major whey proteins constitute approximately 95% and 80% of total whey proteins in colostrum and mature milk, respectively. The remaining protein fraction includes a multitude of proteins that may play important physiological roles in breast fed infants. Characterization of these proteins is necessary to further correlate the protein fraction with physiological and nutritional properties associated with human milk. The technique described here, based on immunoaffinity chromatography, allows isolation of the minor protein fraction, which can be followed by further characterization of the proteins. ACKNOWLEDGMENTS This project was supported in part by NIH Contract HD-6-2923.

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L.R. WOODHOUSEand B. LONNERDAL

REFERENCES 1.

Ogra PL, Greene HL. Human milk and breastfeeding: the art. Pediatr. Res. 1982; ]6:266-71.

an update on the state of

2.

Kovar MG, Serdula MK, Marks JS, Fraser, DW. Review of the epidemiologic evidence for an association between infant feeding and infant health. Pediatrics suppl. 1984: 615-38.

3.

Blanc B. Biochemical aspects of human milk - comparison with bovine milk. Wld. Rev. Nutr. Diet 1981; 36:]-89.

4.

Ribadeau-Dumas B. Human milk. Endeavor 1983; 7:80-7.

5.

Butler JE. Immunologic aspects of breast feeding, anti-infectious activity of breast milk. Semin. Perinatol. 1979; 33:255-70.

6.

Gaull GE, Wright CE, Isaacs CE. Significance of growth modulators in human milk. Pediatrics 1985; 75:142-5.

7.

Hambraeus L, LSnnerdal B, Forsum E, Gebre-Medhin M. Nitrogen and protein components of human milk. Acta Paediatr. Scand. 1978; 67:561-5.

8.

Bezkorovainy A. Human milk and colostrum proteins: a review. J. Dairy Sci. 1977; 60:1023-37.

9.

Anderson NG, Powers MT, Tollaksen SL. Proteins of human milk. fication of major components. Clin. Chem. 1982; 28:1045-55.

I. Identi-

10.

LSnnerdal B. Biochemistry and physiological functions of human milk proteins. Am. J. Clin. Nutr. 1985; 42:1299-1317.

11.

Gaull GE, Jensen RG, Rassin DK, Malloy MH. Human milk as food. Adv. Perinat. Med. 1982; 2:47-120.

12.

Shahani KM, Kwan AJ, Friend BA. Role and significance of enzymes in human milk. Am. J. Clin. Nutr. 1980; 33:1861-8.

13.

Hansen S], Holm J, Lyngbye J. Folate binding by human milk protein. Scand. J. Clin. Lab. Invest. 1977; 37:363-7.

14.

Samson RR, McClelland DBL. Vitamin B-J2 in human colostrum and milk. Acta Paediatr. Scand. 1980; 69:93-9.

I5.

Hollis BW, Draper HH. A comparative study of vitamin D-binding globulins in milk. Comp. Biochem. Physiol. 1979; 64B:41-6.

16.

Payne DW, Peng L-H, Pearlman WH, Talbert LM. Corticosteroid-binding proteins in human colostrum and milk and rat milk. J. Biol. Chem. 1976; 251:5272-9.

17.

Goldman AS, Garza C, Nichols BL, Goldblum RM. Immunologic factors in human milk during the f i r s t year of lactation. J. Pediatr. 1982; 100:563-7.

18.

Parry RM, Chandan RC, Shahani KM. Isolation and characterization of human milk lysozyme. Arch. Biochem. Biophys. 1969; 103:59-65.

ISOLATION OF HUMANWHEYPROTEINS

863

19.

Underdown BJ, DeRose J, Koczekan D, Socken D, Weicker J. Isolation of human secretory component by a f f i n i t y chromatography on IgM-Sepharose. ImmUnochemistry 1977; 14:111-8.

20.

Brown WR, Newcomb RW, Ishizaki K. Proteolytic degradation of exocrine and serum immunoglobulins. J.iClin. Invest. 1970; 49:1374-80.

21.

Underdown BJ, Dorrington KJ. Studies on the structural and conformational basis for the relative resistance of serum and secretory immunoglobulin A to proteolysis. J. Immunol. 1974; 112:949-59.

22.

Lindh E. Increased resistance of immunoglobulin A dimers to proteolytic degradation after bindings of secretory component. J. Immunol. 1975; 114:284-6.

?3.

Brandtzaeg P. Mucosa! and glandular distribution of immunoglobulin components: differential localization of free and bound SC in secretory epithelial cells. J. Immunol. 1974; 112:1553-9.

24.

KobByashi K, Vaerman JP, Heremans JF. A facile method to isolate J-chain from polymeric serum IgA or IgM. Immunochemistry 1974; 11:169-71.

25.

Hernell O, Blackberg L, Fredrikzon B, Olivecrona T. Bile salt-stimulated lipase in human milk and lipid digestion during the neonatal period. In: Lebenthal E, ed. Textbooklof Gastroenterology and Nutrition in Infancy. New York: Raven Press, 1981: 465-71.

26.

Hambraeus L, Forsum E, Abrahamsson L, LSnnerdal B. Automatic total nitrogen analysis in nutritional evaluation using a block digestor. Anal. Biochem. 1976; 72:78-85.

27.

Bailey GS. Immunodiffusion in gels. In: Walker JM, ed. Methods in Molecular Biology, vol. I, Proteins. Clifton, NJ: The Humana Press, Inc., 1984: 301-10.

28. Walker JM Rocket immunoelectrophoresis. In: Walker JM, ed. Methods in Molecular Biology, vol. 1, Proteins. Clifton, NJ: The Humana Press, Inc., 1984: 3]7-23. 29.

Laurell CB, McKay EJ. Electroimmunoassay. Methods Enzymol. 1981; 73:339-69.

30.

Davidson LA, LSnnerdal B. Isolation and characterization of Rhesus monkey milk lactoferrin. Pediatr. Res. 1985; 20:197-201.

31.

Vaerman JP. Single radial immunodiffusion. Methods Enzymol. 1981; 73:291-305.

32.

LSnnerdal B, Glazier C. Calcium binding by ~-lactalbumin in human milk. J. Nutr. 1985; 115:1209-16,

3.

L~nnerdal B, tSas T. Improved agarose for immunoelectrophoresis. Anal. Biochem. 1976; 72:527-32.

34.

Weeke B. General remarks on principles, equipment, reagents and procedures. Scand. J. Immunol. 1973; 2(suppl 1):15-36.

35.

Mayes ELV Immunoaffinity purification of protein antigens. In: Walker JM, ed. Methods in Molecular Biology, vol. 1, Proteins. Clifton, NJ: The Humana Press, Inc., 1984:13-20.

864

L.R. WOODHOUSEand B. LONNERDAL

36.

Pharmacia Fine Chemicals. A f f i n i t y Chromatography - Principles and Methods. LjungfSretagen AB, Orebro, Sweden, 1983.

37.

Smith BJ. Acetic acid-urea polyacrylamide gel electrophoresis of proteins. In: Walker JM, ed. Methods in Molecular Biology, vol. I , Proteins. C l i f t o n , NJ: The Humana Press, Inc., 1984: 63-73.

38.

LSnnerdal B, Forsum E, Hambraeus L. The protein content of human milk. I. A transversal study of Swedish normal material. Nutr. Rep. I n t . 1976; 13:125-34.

Accepted for publication January 2, 1988